This invention relates to novel bio chemical compounds and compositions having pharmaceutical and medical activities, and to processes for their preparation and use. More specifically, it relates to novel peptide compounds and compositions of the histatin type, which exhibit antimicrobial properties.
Antimicrobial peptides have been found in a variety of organisms. Among the most studied are the magainins from the skins of amphibians, the cecropins from the moth cecropia, melittin and bombolitin from bee venom. In all cases these are cationic peptides reportedly exerting their antibacterial action by altering the membrane permeability. These peptides have an amphiphatic xcex1-helical structure which is critical for their activity (1). Another class of potent antimicrobial peptides, the defensins, was isolated from mammalian phagocytic cells. Defensins have a broad range of activity against bacteria, fungi, viruses and tumour cells and are considered to be important in the host non-immune defense system. X-ray and NMR analysis has revealed that the defensins assume an antiparallel xcex2-sheet conformation. It has also been shown that defensins form voltage dependent channels in model lipid bilayer.
Another class of antimicrobial peptides was recently identified in human saliva. They were originally called histidine-rich peptides (HRP)(2) and later referred to as histatins (3). Twelve members of this family have been characterized (3,4). Histatins are secreted by the parotid and submandibular glands and constitute 3 percent dry weight of saliva (2). The three major forms are histatin-1 (H-1), histatin-3 (H-3), and histatin-5 (H-5) which are 38, 32 and 24 amino acids peptides, respectively and have highly homologous sequence, as depicted in the accompanying Figure. H-1 and H-3 are derived from different genes (5), whereas H-5 is proteolytically derived from H-3. Other members of this family are proteolytically produced from the three main forms although the enzyme(s) responsible for this processing is presently unknown. The smaller members have reduced antimicrobial activities. H-1, H-3 and H-5 contain several basic residues including seven conserved histidines. Homologous histatin sequences have also been isolated from the saliva of other primates.
These peptides are highly basic (cationic), non-amphiphilic and show no homology to other known proteins. Histatins have been implicated in several biological functions. Histatin-3 and -5 possess antibacterial and antifungal properties at physiological concentrations and may play an important role in the non-immune oral host defense system.
Histatins have been shown to have a number of different biological activities. A phosphorylated member, histatin-1, selectively adsorbs to hydroxylapatite and enamel powder, possibly acting as a precursor of the acquired enamel pellicule (6). More recently, H-5 has been shown to act synergistically with epidermal growth factor (EGF) to promote rabbit chondrocyte proliferation (7). Patents describing the use of histatins for wound healing, and bond and periodontal tissue regeneration have also appeared (8-10).
It has been reported that H-1, H-3 and H-5 possess potent antimicrobial properties against bacteria (11) and fungi (12) in a concentration dependent manner at physiological concentrations. The most potent, H-5, has a minimum inhibitory concentration (MIC) of 10 xcexcg/ml against C. albicans (12). This value compares favorably with the widely used antifungal agent ketoconazole, which is an inhibitor of lanosterol C-14 demethylase, an important enzyme in the biosynthesis of ergosterol (13). The mode of action of histatin is still unknown.
In the one of the earliest structure-activity relationship (SAR) studies of histatins published to date, Raj et al. reported that the C-terminal fragment is most important and that a minimum of 16 amino acids (H-58-24) were required for activity against C. albicans (14). Because of the large number of positive charge on H-58-24, these authors suggested that histatins interact with the polar head groups of the phospholipid membranes, but only weak experimental evidence supports this hypothesis. Raj and co-workers also showed by CD and NMR that H-58-24 has no structure in H2O but adopts an extended xcex1-helical conformation in trifluoroethanol (TFE) or in DMSO (14). The same group also reported that the H-58-24 fragment has xcex1-helical character in the presence of DMPC vesicles, and that the conformation is pH independent (15). More recenty. Oppenheim and co-workers (16) have produced by recombinant methods an H-3 peptide mutant in which the central region of H-3(13-24-13-24) was tandemly repeated. At very low concentrations, this H-3(13-24-13-24) fusion peptide was apparently more active than H-3 in the candicidal assay (16).
The conformation of histatin-5 in water or in the presence of its biological targets has not been firmly established.
It is an object of the present invention to provide novel histatin analogs having improved microbiocidal properties.
The present invention derives from our discoveries that the most bioactive conformation of histatin peptides involves their adoption of a hairpin loop conformation in the region of positions 8-24 of H-5, likely around E16-K17 and that histatin peptides are reasonably tolerant to amino acid replacement. Accordingly, we have found that the introduction of substantially permanent loop structures into H-5 peptide compounds, to provide cyclic analogs of histatins, leads to compounds having much more potent activity than H-5 against a variety of different microorganisms. Since some amino acid replacement can be tolerated in H-5 without significant loss of activity, the introduction of replacement amino acids at appropriate locations in the chain so as to form chemical bridges between themselves (e.g. disulphide bonds between introduced cysteine residues) can be accomplished. In this way, the loop or cyclic structure is substantially permanently imparted to the compounds, subject only to the normal chemical stability of the loop-forming bonds between the replacement amino acids (mutations). The result is a family of novel cyclic peptides analogous to H-5, but having much greater antimicrobial activity than H-5, as set out in the specific examples below.
Thus according to the present invention, from one aspect, there is provided cyclic analogues of histatin H-5 having from about 7-20 amino acid units exhibiting substantial homology to histatin H-5, and having a cyclic portion of from 5-16 of said amino acid units.
The bioactive conformation of H-3 is not an extended helix as inferred by previous reports, but a loop or cyclic structure. Although this loop structure motif is unusual for antimicrobial peptides, it has been observed in brevenins from frog skin or of bactenecins from bovine neutrophils (17).